Helena F. Schteingart

SOX9 and SF1 are involved in cyclic AMP-mediated upregulation of anti-Müllerian gene expression in the testicular prepubertal Sertoli cell line SMAT1

In Sertoli cells, anti-Müllerian hormone (AMH) expression is upregulated by FSH via cyclic AMP (cAMP), although no classical cAMP response elements exist in the AMH promoter. The response to cAMP involves NF-κB and AP2; however, targeted mutagenesis of their binding sites in the AMH promoter do not completely abolish the response. In this work we assessed whether SOX9, SF1, GATA4, and AP1 might represent alternative pathways involved in cAMP-mediated AMH upregulation, using real-time RT-PCR (qPCR), targeted mutagenesis, luciferase assays, and immunocytochemistry in the Sertoli cell line SMAT1. We also explored the signaling cascades potentially involved. In qPCR experiments, Amh, Sox9, Sf1, and Gata4 mRNA levels increased after SMAT1 cells were incubated with cAMP. Blocking PKA abolished the effect of cAMP on Sox9, Sf1, and Gata4 expression, inhibiting PI3K/PKB impaired the effect on Sf1 and Gata4, and reducing MEK1/2 and p38 MAPK activities curtailed Gata4 increase. SOX9 and SF1 translocated to the nucleus after incubation with cAMP. Mutations of the SOX9 or SF1 sites, but not of GAT4 or AP1 sites, precluded the response of a 3,063-bp AMH promoter to cAMP. In conclusion, in the Sertoli cell line SMAT1 cAMP upregulates SOX9, SF1, and GATA4 expression and induces SOX9 and SF1 nuclear translocation mainly through PKA, although other kinases may also participate. SOX9 and SF1 binding to the AMH promoter is essential to increase the activity of the AMH promoter in response to cAMP.

The prepubertal testis: biomarkers and functions.

Purpose of reviewBiomarkers of prepubertal testicular function have become widely available only in recent years. The aim of this review is to update the knowledge on key biomarkers used to assess hypogonadism in boys. Recent findingsSertoli cells are the most representative cells of the prepubertal testis. Anti-Müllerian hormone and inhibin B are essential biomarkers of Sertoli cell function. Also, INSL3 arises as an additional marker of Leydig cell dysfunction. SummaryThe widespread use of these biomarkers has enhanced our knowledge on the pathophysiology and diagnosis of prepubertal male hypogonadism. Beyond their well known germ-cell toxicity, oncologic treatments may also affect Sertoli cell function. Pathophysiology is not the same in all aneuploidies leading to infertility: while hypogonadism is not evident until mid-puberty in Klinefelter syndrome, it is established in early infancy in Down syndrome. In Noonan syndrome, the occurrence of primary hypogonadism depends on the existence of cryptorchidism, and Prader–Willi syndrome may present with either primary or combined forms of hypogonadism. Prepubertal testicular markers have also provided insights into the effects of environmental disruptors on gonadal function from early life, and helped dissipate concerns about testicular function in boys born preterm or small for gestational age or conceived by assisted reproductive technique procedures.

Spreading the Clinical Window for Diagnosing Fetal-Onset Hypogonadism in Boys

In early fetal development, the testis secretes – independent of pituitary gonadotropins – androgens and anti-Müllerian hormone (AMH) that are essential for male sex differentiation. In the second half of fetal life, the hypothalamic–pituitary axis gains control of testicular hormone secretion. Follicle-stimulating hormone (FSH) controls Sertoli cell proliferation, responsible for testis volume increase and AMH and inhibin B secretion, whereas luteinizing hormone (LH) regulates Leydig cell androgen and INSL3 secretion, involved in the growth and trophism of male external genitalia and in testis descent. This differential regulation of testicular function between early and late fetal periods underlies the distinct clinical presentations of fetal-onset hypogonadism in the newborn male: primary hypogonadism results in ambiguous or female genitalia when early fetal-onset, whereas it becomes clinically undistinguishable from central hypogonadism when established later in fetal life. The assessment of the hypothalamic–pituitary–gonadal axis in male has classically relied on the measurement of gonadotropin and testosterone levels in serum. These hormone levels normally decline 3–6 months after birth, thus constraining the clinical evaluation window for diagnosing male hypogonadism. The advent of new markers of gonadal function has spread this clinical window beyond the first 6 months of life. In this review, we discuss the advantages and limitations of old and new markers used for the functional assessment of the hypothalamic–pituitary–testicular axis in boys suspected of fetal-onset hypogonadism.

Bisphenol A effect on glutathione synthesis and recycling in testicular Sertoli cells

Background and objective: Controversial effects of bisphenol A (BPA) have been reported on testicular function. These differences might reflect dissimilar exposure conditions. Dose responses to toxicants may be non-linear, e.g. U-shaped, with effects at low and at high levels of exposure and lower or inexistent effects at intermediate levels. Sertoli cells produce high levels of glutathione (GSH) as a cell defense mechanism. In this study, we addressed the question whether the exposure to different doses of BPA could influence Sertoli cell GSH synthesis and recycling. Materials and methods: Primary Sertoli cell cultures were exposed to various doses of BPA (0.5 nM–100 μM). Cell viability was measured as an outcome of toxic effect. GSH cell content was determined to evaluate cell response to toxicant exposure. Glutamate-cysteine ligase catalytic (GCLC) and modulatory (GCLM) subunit expression were assessed to estimate GSH synthesis, and GSH reductase (GR) expression to estimate GSH recycling. Results: BPA 100 μM, but not lower doses, decreased cell viability. BPA 10 and 50 μM, but not lower doses, induced an increment in Sertoli cell GSH levels, due to a rapid upregulation of GCLC and GR and a slower upregulation of GCLM. Conclusions: High doses of BPA are deleterious for Sertoli cells. Intermediate doses do not affect Sertoli cell viability and increase cell content of GSH owing to increased GSH synthesis and recycling enzyme expression. Lower doses of BPA are not capable of eliciting a cell defense response. These observations may explain a non-linear dose response of Sertoli cells to BPA.

Mono-(2-ethylhexyl) phthalate (MEHP) affects intercellular junctions of Sertoli cell: A potential role of oxidative stress

We analyzed the potential role of oxidative stress induced by mono (2-ethylhexyl) phthalate (MEHP) in adherent cell junction protein expression of prepubertal rat Sertoli cells (SC) in vitro. Five-day SC cultures were treated with MEHP (200μM) for 24h and compared to cells in basal conditions. Western blot and immunofluorescent (IF) analyses showed that MEHP induced increase of N-cadherin and catenin expression, modifying its distribution. Concomitantly, Cx-43 expression decreased significantly and delocalization of the IF signal for tight junction proteins (occludin, claudin-11 and ZO-1) occurred. Indicative of oxidative stress, MEHP induced in SC an increase of lipoperoxides, a decrease in glutathione (GSH) levels and a concomitant increase in Glutathione S-Transferases (GST) activity. Antioxidant N-acetyl-cysteine (1mM) treatment prevented GSH decrease and N-cadherin and α-catenin up-regulation induced by MEHP. Our data suggest that oxidative stress signaling is a mechanism involved in adherent cell junctions disruption induced by MEHP in SC cultures.

Anti-Müllerian hormone as a marker of steroid and gonadotropin action in the testis of children and adolescents with disorders of the gonadal axis

In pediatric patients, basal testosterone and gonadotropin levels may be uninformative in the assessment of testicular function. Measurement of serum anti-Müllerian hormone (AMH) has become increasingly widespread since it provides information about the activity of the male gonad without the need for dynamic tests, and also reflects the action of FSH and androgens within the testis. AMH is secreted in high amounts by Sertoli cells from fetal life until the onset of puberty. Basal AMH expression is not dependent on gonadotropins or sex steroids; however, FSH further increases and testosterone inhibits AMH production. During puberty, testosterone induces Sertoli cell maturation, and prevails over FSH on AMH regulation. Therefore, AMH production decreases. Serum AMH is undetectable in patients with congenital or acquired anorchidism, or with complete gonadal dysgenesis. Low circulating levels of AMH may reflect primary testicular dysfunction, e.g. in certain patients with cryptorchidism, monorchidism, partial gonadal dysgenesis, or central hypogonadism. AMH is low in boys with precocious puberty, but it increases to prepubertal levels after successful treatment. Conversely, serum AMH remains at high, prepubertal levels in boys with constitutional delay of puberty. Serum AMH measurements are useful, together with testosterone determination, in the diagnosis of patients with ambiguous genitalia: both are low in patients with gonadal dysgenesis, including ovotesticular disorders of sex development, testosterone is low but AMH is in the normal male range or higher in patients with disorders of androgen synthesis, and both hormones are normal or high in patients with androgen insensitivity. Finally, elevation of serum AMH above normal male prepubertal levels may be indicative of rare cases of sex-cord stromal tumors or Sertoli cell-limited disturbance in the McCune Albright syndrome.

Androgens downregulate anti-Müllerian hormone promoter activity in the Sertoli cell through the androgen receptor and intact steroidogenic factor 1 sites†

Abstract Testicular anti-Müllerian hormone (AMH) production is inhibited by androgens around pubertal onset, as observed under normal physiological conditions and in patients with precocious puberty. In agreement, AMH downregulation is absent in patients with androgen insensitivity. The molecular mechanisms underlying the negative regulation of AMH by androgens remain unknown. Our aim was to elucidate the mechanisms through which androgens downregulate AMH expression in the testis. A direct negative effect of androgens on the transcriptional activity of the AMH promoter was found using luciferase reporter assays in the mouse prepubertal Sertoli cell line SMAT1. A strong inhibition of AMH promoter activity was seen in the presence of both testosterone and DHT and of the androgen receptor. By site-directed mutagenesis and chromatin immunoprecipitation assays, we showed that androgen-mediated inhibition involved the binding sites for steroidogenic factor 1 (SF1) present in the proximal promoter of the AMH gene. In this study, we describe for the first time the mechanism behind AMH inhibition by androgens, as seen in physiological and pathological conditions in males. Inhibition of AMH promoter activity by androgens could be due to protein–protein interactions between the ligand-bound androgen receptor and SF1 or by blockage of SF1 binding to its sites on the AMH promoter. Summary Sentence Androgens act directly on prepubertal Sertoli cells to inhibit AMH promoter activity in the presence of the androgen receptor and intact SF1 sites.